Chemistry and Chemical Biology ETDs

CHEN DANQI

Publication Date

2-8-2011

Abstract

My doctoral research primarily focuses on two hotdog-fold thioesterases, EntH (also known as YbdB) from E. coli, and BFIT2 from Homo sapiens. The EntH (YbdB) gene is included in a large gene cluster that encodes the enzymes of the biosynthetic pathway leading to enterobactin. Building on the hypothesis that EntH might function in a house-keeping' role by liberating misacylated EntB, two potential pathways to EntB misacylation were identified, one involving the phosphopantetheinyl transferase EntD and the other involving 2,3-DHB-AMP ligase EntE. EntH displays thioesterase activity towards a variety of acyl and aryl-holo EntB adducts. Lastly, It was shown that EntF acts on the 2,3-DHB-holo-EntB quickly, but not quickly on misacylated EntB adducts.tandem hotdog-fold thioesterase domains and a C-terminal steroidogenic acute regulatory protein related lipid transfer (START) domain. The expression of BFIT2 is induced during the thermogenesis transition of brown fat tissue. The expression of the recombinant BFIT2 in transfected HEK cells was confirmed by Western blot analysis. The recombinant BFIT2 contains a N-terminal His6-tag and epitope, which was found to be susceptible to posttranslational removal. The recombinant N-terminal (minus residues 1-34) truncated mutant was found not to undergo posttranslational cleavage, thus suggesting that the N-terminal region is a signal sequence. A chimeric protein BFIT2 N(1-42)-GFP was shown by confocal microscopy to co-locate with the mitochondria. The BFTI2 precursor was shown to be taken up by freshly isolated HEK cell mitochondria and cleaved to the mature form. These results confirmed that the N-terminal region of BFIT2 functions as MTS. During the thermogenesis transition of brown fat tissue, BFIT2 might function to restore the balance between free CoA and fatty acyl-CoA by hydrolyzing the long to medium chain fatty acyl-CoAs. Consistent with this hypothesis, BFIT2 was found to be much more active towards palmitoyl-CoA, myristoyl-CoA and lauroyl-CoA.'

Project Sponsors

NIH

Language

English

Keywords

Function Hotdog Thioesterase

Document Type

Dissertation

Degree Name

Chemistry

Level of Degree

Doctoral

Department Name

Department of Chemistry and Chemical Biology

First Committee Member (Chair)

Mariano, Patrick

Second Committee Member

Allen, Karen

Third Committee Member

Melancon, Charles

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